Confocal laser scanning microscopy, a new in vivo diagnostic tool for schistosomiasis

Background

The gold standard for the diagnosis of schistosomiasis is the detection of the parasite''s characteristic eggs in urine, stool, or rectal and bladder biopsy specimens. Direct detection of eggs is difficult and not always possible in patients with low egg-shedding rates. Confocal laser scanning microscopy (CLSM) permits non-invasive cell imaging in vivo and is an established way of obtaining high-resolution images and 3-dimensional reconstructions. Recently, CLSM was shown to be a suitable method to visualize Schistosoma mansoni eggs within the mucosa of dissected mouse gut. In this case, we evaluated the suitability of CLSM to detect eggs of Schistosoma haematobium in a patient with urinary schistosomiasis and low egg-shedding rates.

Methodology/Principal Findings

The confocal laser scanning microscope used in this study was based on a scanning laser system for imaging the retina of a living eye, the Heidelberg Retina Tomograph II, in combination with a lens system (image modality). Standard light cystoscopy was performed using a rigid cystoscope under general anaesthesia. The CLSM endoscope was then passed through the working channel of the rigid cystoscope. The mucosal tissue of the bladder was scanned using CLSM. Schistoma haematobium eggs appeared as bright structures, with the characteristic egg shape and typical terminal spine.

Conclusion/Significance

We were able to detect schistosomal eggs in the urothelium of a patient with urinary schistosomiasis. Thus, CLSM may be a suitable tool for the diagnosis of schistosomiasis in humans, especially in cases where standard diagnostic tools are not suitable.     

Rod photoreceptor ribbon synapses in DBA/2J mice show progressive age-related structural changes

The DBA/2J mouse is a commonly used animal model in glaucoma research. The eyes of DBA/2J mice show severe age-related changes that finally lead to the degeneration of retinal ganglion cells and the optic nerve. Recent electroretinogram studies identified functional deficits, which suggest that also photoreceptor cells are involved in the pathological processes occurring in the DBA/2J mouse retina. In a comparative study, we examined anatomical and molecular changes in the retinae of DBA/2J and C57BL/6 control mice with light and electron microscopy and with PCR analyses. In the retina of the DBA/2J mouse, we found a thinning of the outer plexiform layer, the first synaptic layer in the transfer of visual signals, and age-dependent and progressive degenerative structural changes at rod photoreceptor ribbon synapses. The structural ribbon changes represent a photoreceptor synaptic phenotype that has not yet been described in this animal model of secondary angle-closure glaucoma. Furthermore, genes of the classical complement cascade were upregulated in the photoreceptor cells of aging DBA/2J mice, suggesting a putative link between ribbon synapse degradation and the innate immune system.     

Job strain and tobacco smoking: an individual-participant data meta-analysis of 166,130 adults in 15 European studies

Background

Tobacco smoking is a major contributor to the public health burden and healthcare costs worldwide, but the determinants of smoking behaviours are poorly understood. We conducted a large individual-participant meta-analysis to examine the extent to which work-related stress, operationalised as job strain, is associated with tobacco smoking in working adults.

Methodology and Principal Findings

We analysed cross-sectional data from 15 European studies comprising 166 130 participants. Longitudinal data from six studies were used. Job strain and smoking were self-reported. Smoking was harmonised into three categories never, ex- and current. We modelled the cross-sectional associations using logistic regression and the results pooled in random effects meta-analyses. Mixed effects logistic regression was used to examine longitudinal associations. Of the 166 130 participants, 17% reported job strain, 42% were never smokers, 33% ex-smokers and 25% current smokers. In the analyses of the cross-sectional data, current smokers had higher odds of job strain than never-smokers (age, sex and socioeconomic position-adjusted odds ratio: 1.11, 95% confidence interval: 1.03, 1.18). Current smokers with job strain smoked, on average, three cigarettes per week more than current smokers without job strain. In the analyses of longitudinal data (1 to 9 years of follow-up), there was no clear evidence for longitudinal associations between job strain and taking up or quitting smoking.

Conclusions

Our findings show that smokers are slightly more likely than non-smokers to report work-related stress. In addition, smokers who reported work stress smoked, on average, slightly more cigarettes than stress-free smokers.     

RNF8 and RNF168 but not HERC2 are required for DNA damage-induced ubiquitylation in chicken DT40 cells

The ubiquitylation cascade plays an important role in the recruitment of repair factors at DNA double-strand breaks. The involvement of a growing number of ubiquitin E3 ligases adds to the complexity of the DNA damage-induced ubiquitin signaling. Here we use the genetically tractable avian cell line DT40 to investigate the role of HERC2, RNF8 and RNF168 in the DNA damage-induced ubiquitylation pathway. We show that formation of ubiquitin foci as well as cell survival after DNA damage depends on both RNF8 and RNF168. However, we find that RNF8 and RNF168 knockout cell lines respond differently to treatment with camptothecin indicating that they do not function in a strictly linear manner. Surprisingly, we show that HERC2 is required neither for survival nor for ubiquitin foci formation after DNA damage in DT40. Moreover, the E3 ubiquitin ligase activity of HERC2 is not redundant to that of RNF8 or RNF168.     

Multi-substrate flavonol O-glucosyltransferases from strawberry (Fragaria x ananassa) achene and receptacle

In an effort to characterize fruit ripening-related genes functionally, two glucosyltransferases, FaGT6 and FaGT7, were cloned from a strawberry (Fragaria x ananassa) cDNA library and the full-length open reading frames were amplified by rapid amplification of cDNA ends. FaGT6 and FaGT7 were expressed heterologously as fusion proteins in Escherichia coli and target protein was purified using affinity chromatography. Both recombinant enzymes exhibited a broad substrate tolerance in vitro, accepting numerous flavonoids, hydroxycoumarins, and naphthols. FaGT6 formed 3-O-glucosides and minor amounts of 7-O-, 4'-O-, and 3'-O-monoglucosides and one diglucoside from flavonols such as quercetin. FaGT7 converted quercetin to the 3-O-glucoside and 4'-O-glucoside and minor levels of the 7- and 3'-isomers but formed no diglucoside. Gene expression studies showed that both genes are strongly expressed in achenes of small-sized green fruits, while the expression levels were generally lower in the receptacle. Significant levels of quercetin 3-O-, 7-O-, and 4'-O-glucosides, kaempferol 3-O- and 7-O-glucosides, as well as isorhamnetin 7-O-glucoside, were identified in achenes and the receptacle. In the receptacle, the expression of both genes is negatively controlled by auxin which correlates with the ripening-related gene expression in this tissue. Salicylic acid, a known signal molecule in plant defence, induces the expression of both genes. Thus, it appears that FaGT6 and FaGT7 are involved in the glucosylation of flavonols and may also participate in xenobiotic metabolism. The latter function is supported by the proven ability of strawberries to glucosylate selected unnatural substrates injected in ripe fruits. This report presents the first biochemical characterization of enzymes mainly expressed in strawberry achenes and provides the foundation of flavonoid metabolism in the seeds.     

Invagination of presynaptic ribbons in the fly's optic lobe following loss of their target neuron.

In the first optic neuropile of the housefly Musca, photoreceptor terminals innervate fixed clusters of interneurons, one of which is the monopolar cell L2; L2's synapses in turn feed back upon the terminals. We examined the ultrastructure of these feedback synapses following degeneration of their normal targets, the receptor terminals; this was accomplished by photo-ablating the receptor cells after intraretinal injections of sulforhodamine. Even when all the terminals degenerated, their deafferentated target cells, including L2, remained structurally intact for at least 14 d. Despite this lack of obvious trans-synaptic degeneration, L2's synaptic connections did alter. Presynaptic organelles of the feedback synapses, synaptic ribbons and associated synaptic vesicles, soon appeared in L2's cytoplasm, separating from their site of attachment at the presynaptic membrane by invagination. Similar free-floating organelles and vesicles also occurred in another monopolar cell, L4. They were also occasionally encountered in L2, in normal, newly emerged flies at a time when a naturally occurring loss of feedback synapses is greatest. We interpret the process of internalization that forms these floating ribbons to be the first step in synaptic loss which occurs spontaneously, and that the rate is enhanced in L2 when its main synaptic targets, the receptor terminals, degenerate.